Tick-borne bacteria in the family Anaplasmataceae cause emerging zoonoses across the globe, such as human anaplasmosis and ehrlichioses, where competent vector ticks occur. Due to the non-specific signs and symptoms they cause, and absence of diagnostic antibodies at the time of illness onset, they may be confused with better recognized febrile illnesses such as rickettsioses or viral diseases. Severe human ehrlichiosis is characterized by a pro- inflammatory syndrome, but the bacterial gene products responsible for inducing adverse host reactions are unknown. Determination of gene function in the Anaplasmataceae is complicated by their obligately intracellular nature, yet, this would allow identification of targets for prevention and treatment of disease. Previous animal models of ehrlichiosis do not reproduce features of human disease, which makes it difficult to investigate virulence factors. Our group has isolated a novel human ehrlichiosis agent (Ehrlichia muris-like agent, EMLA) in vitro from both a human patient and a blacklegged tick. EMLA causes disease in laboratory mice that recapitulates human monocytic ehrlichiosis, making it now possible to investigate the factors that cause clinical ehrlichiosis in a mouse model. To elucidate the molecular basis of pathogenicity and tick transmission in ehrlichiae, we propose to generate a library of mutant EMLA using random mutagenesis and characterization by Illumina sequencing-based insertion site determination. We will use a transposon containing a cassette that can subsequently be replaced with a functional gene copy for complementation. We will implement our plan through completion of the following specific aims: AIM 1: Produce a library of Ehrlichia muris-like agent (EMLA) mutants with a replaceable selection cassette in tick and human cell cultures. AIM 2a: Map insertion sites of transposons by Illumina-based sequence analysis of mutant pools followed by assignment to individual mutant lines using PCR. 2b. Identification of mutants defective for invasion and successful colonization of human and tick cells using cell invasion/colonization screens, bioinformatics analysis, and genetic complementation. Ehrlichia species differentially express genes depending on the host cell in which they reside. We will raise mutants in human and tick cells to recover bacteria with disrupted genes that are essential for colonization of only one host cell type, as well as those that govern pathogenicity but are not essential. We realize that genes required for colonization of and transmission by ticks are important for ability of ehrlichiae to infect mammals. With the availability of the human, the EMLA, and the Ixodes scapularis genomes, the conditions are now right to identify ehrlichia genes that mediate interactions with its host and vector in vitro, setting the stage for more comprehensive studies in a subsequent project.